bilirubin .ppt.. dr.anand r

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BILIRUBIN METABOLISM &INHERITED DISORDERS OF BILIRUBIN METABOLISM : 

BILIRUBIN METABOLISM &INHERITED DISORDERS OF BILIRUBIN METABOLISM Dr.ANAND R

CRUNCHES…. : 

CRUNCHES…. 80% bilirubin originates from senescent RBCs 1-2 х 10*8 RBCs destroyed/hour 6g hb produced in body/day 250-300 mg bilirubin produced/day

Pathway for RBC Scavenging : 

Pathway for RBC Scavenging Liver, Spleen & Bone marrow Hemoglobin Globin Amino acids Amino acid pool Heme Bilirubin Fe2+ Excreted Phagocytosis & Lysis Through Liver 5

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Bilirubin synthesis

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Heme oxygenase (HO)- 3 isoforms HO-1 : Inducible form HO-2 : Constitutive form HO-3 : Minor form Requires a metal and Oxygen for activation Iron in heme binds Oxygen –reductive activation

Clinical significance: : 

Clinical significance: Non iron metalloporphyrins- tin and zinc protoporphyrin , used as treatment in hyperbilirubinemia Binds HO-2 with higher affinity,doesn’t activate Dead end inhibitor of HO-2

CHEMISTRY OF BILIRUBIN : 

CHEMISTRY OF BILIRUBIN 1’8’-Dioxo-1,3,6,7- tetramethyl-2,8-divinylbiladiene-a,c-dipropionic acid

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Bilirubin insolubility- a paradox Insoluble despite of presence of 2 propionic acid side chains,4 amino groups,2 lactam oxygens `ridge-tile’ configuration 4Z,15Z trans configuration Intra molecular Hydrogen bonding SIGNIFICANCE: Bilirubin conjugation in body Basis of indirect diazo reaction photoisomerisation

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Fog J, Jellum E. Structure of bilirubin.

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Molecular model of bilirubin structure

BILIRUBIN TRANSPORTRole of albumin : 

BILIRUBIN TRANSPORTRole of albumin Unconjugated bilirubin-sparingly soluble in water at physiological pH. In circulation-bound to albumin Purpose: Prevents precipitation and deposition in tissues Aids transport form site of production to site of elimination-liver Prevents bilirubin from entering brain

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Binding sites on albumin: 1ͦ binding site- residues 124-297 lesser extent 446-547 Gitzelmann-Cumarasamy N, Kuenzle CC, Wilson KJ. Mapping 1976 Lysine 240-proposed to be site of high affinity binding Jacobsen C:Biochem J 1978; 171:453. Unconjugated bilirubin binding –reversible Conjugated bilirubin can bind covalently- delta bilirubin Clinical significance: Other ligands –drugs can bind to albumin and displace bilirubin

HEPATIC UPTAKE : 

HEPATIC UPTAKE Remains to be identified conclusively Enters hepatocyte across sinusoidal membrane Bilirubin is of enough low polarity – can cross membranes without aid of transporters Zucker SD, Goessling W; Biochim Biophys Acta 2000; 1463:197 Bilirubin uptake- liver specific function A carrier protein thought to be involved- OATP 2/OATP C/ SLC21A6

STORAGE WITHIN HEPATOCYTE : 

STORAGE WITHIN HEPATOCYTE Remains bound to cytosolic proteins Two fractions observed- Y,Z PROTEINS Normal concentrations of bilirubin- bound to Y fraction- predominant proteins Z fraction apparent at high concentrations of bilirubin Ligandin (Y Protein)-binds many other compounds Belong to Glutathine-S-Transferases(GST) family

BILIRUBIN CONJUGATION : 

BILIRUBIN CONJUGATION Essential for biliary excretion of bilirubin Glucuronic acid – major conjugating sugar in bile Glucosyl,xylosyl conjugates-traces Catalyzed by UDP-glucuronosyltransferase(UGT)

UGT ENZYME SYSTEM : 

UGT ENZYME SYSTEM 2 Major families UGT 1: Major bilirubin conjugating form in human UGT 2: Conjugation of steroids,other endogenous and exogenous substrates Multiple isoforms present One isoform of UGT 2 family: inducible by phenobarbital Significance: UGT 1A locus abnormalities- disorders of bilirubin conjugation phenobarbital used as enzyme inducer in some conditions of hyperbilirubinemia

UGT 1 COMPLEX : 

UGT 1 COMPLEX Chromosome 2q37 Exons 2,3,4,5- used in all isoforms expressed from this locus,so mutations affect all isoforms Exons 1A1-12: variable ; each exon has different promoter region and differently regulated,mutation affects only the corresponding isoform

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ATATATATATATATAA ATATATA(TA)TATATATAA

BILIRUBIN EXCRETION : 

BILIRUBIN EXCRETION The rate limiting step in bilirubin metabolism Excreted across canalicular membrane into bile MRP(Multidrug resistant protein) family – efflux pumps MRP 2-essential for bilirubin excretion Significance: Deficiency of MRP 2: DUBIN JOHNSON SYNDROME MRP 2- downregulated in cholestasis

SUMMARY OF BILIRUBIN METABOLISM IN LIVER : 

SUMMARY OF BILIRUBIN METABOLISM IN LIVER

BILIRUBIN IN INTESTINE : 

BILIRUBIN IN INTESTINE 23

BILIRUBIN HANDLING IN KIDNEY : 

BILIRUBIN HANDLING IN KIDNEY 24

INHERITED DISORDERS OF BILIRUBIN METABOLISM : 

INHERITED DISORDERS OF BILIRUBIN METABOLISM UNCONJUGATED HYPERBILIRUBINEMIA CRIGLER NAJJAR SYN. TYPE 1 CRIGLER NAJJAR SYN. TYPE 2 (ARIAS SYN.) GILBERT SYNDROME CONJUGATED HYPERBILIRUBINEMIA DUBIN JOHNSON SYNDROME ROTOR SYNDROME

CRIGLER NAJJAR SYNDROME TYPE 1 : 

CRIGLER NAJJAR SYNDROME TYPE 1 Described by Crigler and Najjar in 1952. Autosomal recessive inheritance Incidence 0.6-1.0/ million; occurs in all races Characterised by striking unconjugated hyperbilirubinemia 20-45 mg/dL (340-765 µmol/L) Jaundice Appears in neonatal period ; persists for life

MOLECULAR MECHANISM : 

MOLECULAR MECHANISM TYPE 1A : Defect in conjugation of various drugs and xenobiotics in addition to bilirubin Constitutes majority of cases; mutations in exons 2-5 TYPE 1B : Defect limited to bilirubin conjugation Small subset of patients; mutations in exon A1

CLINICAL FEATURES : 

CLINICAL FEATURES Severe unconjugated hyperbilirubinemia at birth Prior to phototherapy: Kernicterus Death in infancy

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With phototherapy: Child may survive infancy Jaundice persists throught life Prone to kernicterus throught life

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LAB FEATURES: High serum bilirubin 18-30 mg, no evidence of hemolysis Other parameters within normal limits Bilirubin not present in urine; bile lacks glucuronides Liver biopsy shows normal histology Molecular diagnostics for gene sequence abnormalities for diagnosis

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TREATMENT: Aims at reducing bilirubin levels Exchange tranfusion: in immediate neonatal period Phototherapy: Main stay of treatment Special blue lamps used;wavelength 450-500 nm

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Phototherpy for infants

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Becomes less effective near puberty Plasmapheresis: Effective in emergencies Orthotopic liver transplantation: Standard treatment for CN-1

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NOVEL APPROACHES: Inhibition of heme oxygenase: Tried with tin-mesoporphyrin Showed promise in animal studies Effect in humans- variable Bilirubin degradation by bilirubin oxidase: Obtained from myrothecium verrucaria In animal studies Cytochrome p 450 induction: Various inducers available Provides alternate pathway for degradation.

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Isolated liver cell tranplantation: Transplantation of liver cells instead of entire liver Results were not satisfactory Gene therapy: Replacement with normal bilirubin UGT gene- attractive therapeutic modality Approaches under evaluation

CRIGLER NAJJAR SYN.2 : 

CRIGLER NAJJAR SYN.2 ARIAS Syndrome Milder variant Autosomal recessive Differs from CN 1 by: Average bilirubin concentrations low Infrequently associated with kenicterus Bile deeply coloured; bilirubin monoglucuronides present UGT1A activity reduced;not absent totally Bilirubin concentration falls by >25% with phenobarbital administration

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Molecular mechanisms: Characterised by aminoacid substitutions – reduce UGT1A1 activity, not abolish it Clinical features: Asymptomatic in most cases Treatment: Similar to CN-1

GILBERT SYNDROME : 

GILBERT SYNDROME pronounced 'zheel-BAYR', often shortened to GS, also called Gilbert-Meulengrachts syndrome Described by Gilbert in 1901 Most common inherited disorder of bilirubin metabolism Males > females; Prevalence 8-10% Presentation: Bilirubin levels remain < 3mg/DL; may increase during intercurrent illness or stress Jaundice is the only positive finding often Routine lab tests are normal

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Molecular mechanism: ATATATATATATATAA ATATATA(TA)TATATATAA Promoter-reporter studies show that an increased TATAA box length reduces UGT1A1 expression Bosma PJ, Roy Chowdhury J, Bakker C, et al. The genetic basis of the reduced expression of bilirubin UDP- glucuronosyltransferase 1 in Gilbert’s syndrome. N Engl J Med 1995; 333:1171. UGT 1A1*28

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?Defect in bilirubin uptake: decreased clearance may also be related to defective uptake Effect of fasting: 2-3 fold increase in s.bilirubin observed on reducing caloric intake to 400 cal. For 48 hours. Limited use in diagnosis

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Effect of Nicotinic Acid Administration: Intravenous nicotinic acid administration increases unconjugated hyperbilirubinemia, probably by : increasing RBC fragility splenic HO activity splenic bilirubin formation. proposed as a provocative test for the diagnosis of GS.

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DIAGNOSIS: Presumptive diagnosis made if: Mild unconjugated hyperbilirubinemia on several occasions Serum GGT,ALP,fasting and postcibal bile acids normal Confirmation: Estimate relative concentrations of bilirubin monoglucuronide to diglucuronide Genetic analysis

BRAIN TEASER!!!! : 

BRAIN TEASER!!!! Handling of most xenobiotics normal in patients with GS.. But one drug…… Name the drug??

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It’s IRINOTECAN IRINOTECAN Carboxy esterase 2 SN-38 UGT 1A1 SN-38 G ELIMINATED TOXIC EFFECTS

DUBIN JOHNSON SYNDROME : 

DUBIN JOHNSON SYNDROME First described by DUBIN & JOHNSON in 1954 Characterised by chronic non-hemolytic jaundice with accumulation of conjugated bilirubin in serum Dark pigmented liver- dark liver jaundice Autosomal recessive ; both sex affected Clinical features: Mild icterus Asymptomatic Hyperbilirubinemia during illness, pregnancy

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PATHOGENESIS: Defeciency of MRP 2 protein on canalicular membrane. Tm reduced; storage normal

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LAB FEATURES: S.Bilirubin levels 2-5mg/ Dl Other parameters: normal Liver grossly pigmented..

BRAIN TEASER!!! : 

BRAIN TEASER!!! Pigment accumulating in liver in DJS?? Melanin?? No!!! Epinephrine metabolites..

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ctd.. URINE COPROPORPHYRIN EXCRETION: Normally 80% of coproporphrin excreted in urine in coproporphyrin III. In DJS-total coproporphyrin excreted –normal; Over 75% is coproporphyrin I DIAGNOSTIC OF DJS Mechanism behind: not known

ROTOR SYNDROME : 

ROTOR SYNDROME First described by ROTOR in 1948 Rare disorder autosomal recessive inheritance Predominantly conjugated hyperbilirubinemia

ctd.. : 

ctd.. Shares many features with DJS. Liver not pigmented. Both Tm and storage reduced Abnormality: not known

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Lab features: Urine coproporphyrin excretion: Total coproporphyrin excretion 250-300% of normal 50-75% fraction is coproporphyrin I Other parameters: Normal limits Liver histology: normal

BILIRUBIN- FRIEND OR FOE?? : 

BILIRUBIN- FRIEND OR FOE?? Function as natural antioxidants in newborns Attenuates graft rejection in cardiac transplant models Inverse relation between bilirubin and coronary artery disease Inverse relation between bilirubin and colorectal cancer

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OOPS!!! I DON’T ENCOURAGE GIFTS!!  